The long-term objective of this project is to determine the molecular mechanisms by which different MBD proteins target specific methylated DNA sequences to carry out distinct functional roles. In mammals, the MBD protein family consists of at least seven proteins involved in different aspects of normal and pathologic developmental gene silencing, DNA mismatch repair, and aberrant tumor suppressor gene silencing in carcinogenesis. In studying the molecular details of MBD2 bound to methylated DNA, we have shown that the protein can exchange rapidly between binding sites in the same DNA molecular. This observation led us to postulate that the distinct functional roles depend, at least in part, o dynamic and structural DNA binding characteristics of the particular MBD involved. Building on this observation, we plan to investigate the sequence dependent and independent binding of MBDs to DNA in the context of multiple target sites. In particular, we will focus on the structure and binding affinity of MBD2, MBD3, and MBD4. Although MBD2 and MBD3 are highly homologous, MBD2 binds with greater affinity and selectivity for methylated DNA. In contrast, MBD4 recognizes a G-T base pair mismatch arising from hydrolytic deamination of methyl-cytosine. The proposed work will involve NMR spectroscopy, surface plasmon resonance, isothermal titration calorimetry, and fluorescence anisotropy techniques. We will correlate the results of these biophysical and structural analyses with studies in tissue culture cells comparing the function of MBD2 mutants and domain swap chimeras. The latter studies will determine whether functional specificity of different MBD proteins reflects the DNA binding and structural properties of the MBD itself. These experiments will involve co-IP, ChIP, and gene expression studies in tissue culture cells. Ultimately, this work will provide unique insight into the mechanisms of DNA methylation dependent gene silencing and DNA mismatch repair. PUBLIC HEALTH RELEVANCE: Studying the details of how protein complexes bind to DNA and regulate the expression of genes advances our ability to modify the expression of specific genes for therapeutic benefit. In these studies, we will examine the binding characteristics of a family of related proteins, the methyl-cytosine binding domain (MBD) proteins, which recognize methylated DNA and control expression of key genes during development and tumor suppressor genes in cancer. The results of these studies will resolve why different MBD proteins control expression of specific genes and provide a rational basis to design inhibitors of individua MBD proteins for therapy of human disease.